Modification of mouse testicular lactate dehydrogenase by pyridoxal 5'-phosphate.
1. Mouse C4 lactate dehydrogenase treated in the dark with pyridoxal 5'-phosphate at pH8.7 and 25 degrees C loses activity gradually; 1mM-pyridoxal 5'-phosphate causes 83% inactivation, and higher concentrations of the reagent cause no further loss of activity. 2. The final extent of inactivation is very pH-dependent, greater inactivation occurring at the high pH values. 3. Inactivation may be fully reversed by addition of cysteine, or made permanent by reducing the enzyme with NaBH4. 4. The absorption spectrum of inactivated reduced enzyme indicates modification of lysine residues. Inactivation by 80% corresponds to modification of at least 1.8 mol of lysine/mol of enzyme subunit. 5. There is no loss of free thiol groups after inactivation with pyridoxal 5'-phosphate and reduction of the enzyme. 6. NAD+ or NADH gives complete protection against inactivation. protection studies with coenzyme fragments indicate that the AMP moiety is largely responsible for the protective effect. Lactate (10 mM) gives no protection in the absence of added nucleotides, but greatly enhances the protection given by ADP-ribose (1 mM). Thus ADP-ribose is able to trigger the binding of lactate. 7. Pyridoxal 5'-phosphate also acts as a non-covalent inhibitor of mouse C4 lactate dehydrogenase. The inhibition is non-competitive with respect to both NAD+ and lactate. 8. Km values for the enzyme at pH 8.0 and 25 degrees C, with the non-varied substrate saturating, are 0.3 mM-lactate and 5 microM-NAD+. 9. These results are discussed and compared with pyridoxal 5'-phosphate modification of other lactate dehydrogenase isoenzymes and related dehydrogenases.
D000818:Animals; D001665:Binding Sites; D003067:Coenzymes / Q000494:pharmacology; D003545:Cysteine / Q000494:pharmacology; D007527:Isoenzymes; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors*; D007773:Lactates / Q000494:pharmacology; D008239:Lysine; D008297:Male; D051379:Mice; D009711:Nucleotides / Q000494:pharmacology; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D013737:Testis / Q000201:enzymology*
D003067:Coenzymes; D007527:Isoenzymes; D007773:Lactates; D009711:Nucleotides; D011732:Pyridoxal Phosphate; D007770:L-Lactate Dehydrogenase; D008239:Lysine; D003545:Cysteine